The complete chloroplast genome of Holarrhena pubescens and its phylogenetic analysis

Abstract Holarrhena pubescens Wall. ex G. Don, 1837 is an important medicinal plant belonging to the Holarrhena genus in the Apocynaceae family. In this study, the complete chloroplast (cp) genome sequence of H. pubescens was sequenced using the Illumina NovaSeq platform. The cp genome of H. pubescens was 160,108 bp in length with 37.21% overall GC content. The cp genome of H. pubescens containing a large single-copy region (LSC, 88,685 bp), a small single-copy region (SSC, 18,671 bp), and a pair of inverted repeat regions (SSC, 26,376 bp). The cp genome encoded 129 genes, including 84 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Phylogenetic analysis based on complete protein coding genes sequences revealed that H. pubescens was closest to Beaumontia murtonii.


Introduction
Holarrhena pubescens Wall. ex G.Don, 1837 is an important medicinal plant belonging to the Holarrhena genus in the Apocynaceae family (Khan et al. 2021;Namasudra et al. 2021), native to central and southern Africa, the Indian subcontinent, Indochina, and parts of China (Gupta et al. 2021). The bark of H. pubescens has been widely used to treat malaria, cough, cold, bronchopneumonia, dyspepsia, and diarrhea (Sharma et al. 2015;Gupta et al. 2021;Khan et al. 2021;Namasudra et al. 2021). H. pubescens is considered the most valuable medicine due to its numerous medicinal properties and nontoxic side effects (Zahara et al. 2020;Bala et al. 2022). Previous studies have focused on its pharmacological activity and chemical composition (Khan et al. 2021;Sripahco et al. 2021;Chowdhury and Mazumdar 2022). However, there is no report on the genomic studies of H. pubescens. Therefore, we present the complete chloroplast (cp) genome of H. pubescens for the first time, which will provide a theoretical and data foundation for the effective development, protection, and utilization of the H. pubescens. . After the sample is tested as qualified, take 0.5 lg for each sample DNA template, according to the manufacturer's instructions (Illumina NovaSeq 6000), construct a short fragment insertion library (insertion size 350-500 bp) for sequencing, run the pairedend sequencing program (PE), and obtain 150 bp paired-end sequencing reads.

Genome assembly and annotation
The high-quality reads were assembled with GetOrganelle v.1.7.5 (Jin et al. 2020) and annotated by CPGAVAS2

Phylogenetic analysis
To evaluate the phylogenetic position of H. pubescens, 11 complete cp genome sequences from Apocynaceae and one outgroup species (Gentiana crassicaulis) were downloaded from the NCBI database. The complete protein coding genes of 13 species were extracted and aligned by MAFFT v7.471 (Katoh and Standley 2013). Maximum-likelihood (ML) analyses were performed with RAxML v8.2.12 (Stamatakis 2014) using the rapid bootstrap algorithm with 1000 replicates to assess branch support, combined with a search of the best-scoring ML tree under default parameters.

Genome structure analysis
In total, 10.4 Gb of raw data were obtained (69,706,856 reads). The sequences obtained from sequencing are filtered with low quality data to obtain clean reads (67,006,166 reads), and based on the clean reads, an appropriate amount of data is used for assembly and subsequent analysis to finally obtain the complete genome sequence, the genome assembly coverage is 100% ( Figure S1). The complete cp genome of H. pubescens is 160,108 bp in length (Figure 2) and includes a pair of inverted repeats 26,376 bp long, separated by a small and a large single-copy region of 18,671 bp and 88,685 bp, respectively. A total of 129 genes with 84 coding sequences, 37 tRNA genes, and eight unique rRNA sequences were identified. Among them, the ndhD gene is an RNA editing gene, using ACG as the start codon, which was predicted based on the start codon and the length of the gene of the ndhD gene of the closely related species Nerium oleander L. (NC_025656). The GC content of the cp genome was 37.21%.

Phylogenetic analysis
Phylogenetic analysis revealed that H. pubescens was closely related to Beaumontia murtonii Craib (Figure 3). The cp genome sequence of H. pubescens in this study might provide important information for phylogenetic and evolutionary studies in Apocynaceae.